Human leukemia virus-related peptides, antibodies of the peptides and a process for production of the antibodies

ABSTRACT

An antibody of a human leukemia virus-related peptide obtained by collecting an antibody produced in a mammal body by administering to the mammal an antigen prepared by reacting a human leukemia virus-related peptide selected from the group consisting of: 
     (a) a peptide represented by formula (1): 
     
         H-Tyr-Val-Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu-H                (1) 
    
     (b) a peptide represented by formula (2): 
     
         R-Ile-Pro-His-Pro-Lys-Asn-Ser-Ile-Gly-Gly-Glu-Val-OH       (2) 
    
     wherein R is the same as drefined above; 
     (c) a peptide represented by formula (3): 
     
         R-Thr-Trp-Thr-Pro-Lys-Asp-Lys-Thr-Lys-Val-Leu-OH           (3) 
    
     wherein R is the same as defined above; 
     (d) a peptide represented by formula (4): 
     
         H-Val-Val-Gln-Pro-Lys-Lys-Pro-Pro-Pro-Tyr-OH               (4) 
    
     (e) a peptide represented by formula (5): 
     
         R-Met-Gly-Gln-Ile-Phe-Ser-Arg-Ser-Ala-Ser-Pro-OH           (5) 
    
     wherein R is the same as defined above; and 
     (f) a peptide represented by formula (6): 
     
         H-Tyr-Pro-Glu-Gly-Thr-Pro-Lys-Asp-Pro-Ile-Leu-Arg-Ser-Leu-OH (6) 
    
     as a hapten, with a carrier in the presence of a hapten-carrier binding agent.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to novel peptide associated with human leukemia virus (hereafter also referred to as ATLV short for adult T-cell leukemia virus or as HTLV short for human T-cell leukemia virus) and more particularly, to peptides associated with such viral infections as well as mature T-cell leukemia or lymphoma such as adult T-cell leukemia, cutaneous T-cell lymphoma, etc.

DEVELOPMENT OF THE INVENTION

In the specification, amino acids, peptides, protective groups, active groups, nucleotides and others are expressed pursuant to the IUPAC Rules, the IUB Rules or common symbols established in the art when they are abbreviated; examples of which are given below. In case that optical isomers can be present with respect to amino acids or the like, an L-form is meant unless otherwise indicated.

Ser: serine

Leu: leucine

Thr: threonine

Asn: asparagine

Gln: glutamine

Glu: glutamic acid

Lys: lysine

Pro: proline

Val: valine

Trp: tryptophane

His: histidine

Asp: aspartic acid

Gly: glycine

Ile: isoleucine

Ala: alanine

Tyr: tyrosine

Met: methionine

Phe: phenylalanine

Arg: arginine

Cys: cysteine

A: adenine

T: thymine

G: guanine

C: cytosine

Tos: p-toluenesulfonyl group

Boc: tert-butoxycarbonyl group

ONP: p-nitrophenoxy group

Bzl: benzyl group

OBzl: benzyloxy group

Cl₂ -Bzl: 2,6-dichlorobenzyl group

Cl-Z: 2-chlorobenzyloxycarbonyl group

Human leukemia virus has been isolated from a patient with adult T-cell leukemia (ATL) and has been shown to be closely associated with the disease. The provirus gene integrated in host cell DNA was molecularly cloned and the complete nucleotide sequence was determined by M. Yoshida and H. Sugana, the present inventors.

The present invention has been accomplished based on the aforesaid basic information and is directed to such virus-related peptides aiming at diagnosis of such virus infections as well as a process for preparation of and a method of measurement for a specific antibody to these peptides. The nucleotide sequence coding for a precursor of core (gag) proteins of the thus determined virus gene described above is shown in Table 1 below.

                                      TABLE 1                                      __________________________________________________________________________     ATG GGC CAA ATC TTT TCC CGT AGC GCT                                            Met Gly Gln Ile Phe Ser Arg Ser Ala                                            AGC CCT ATT CCG CGA CCG CCC CGG GGG                                            Ser Pro Ile Pro Arg Pro Pro Arg Gly                                            CTG GCC GCT CAT CAC TGG CTT AAC TTC                                            Leu Ala Ala His His Trp Leu Asn Phe                                            CTC CAG GCG GCA TAT CGC CTA GAA CCC                                            Leu Gln Ala Ala Tyr Arg Leu Glu Pro                                            GGT CCC TCC AGT TAC GAT TTC CAC CAG                                            Gly Pro Ser Ser Tyr Asp Phe His Gln                                            TTA AAA AAA TTT CTT AAA ATA GCT TTA                                            Leu Lys Lys Phe Leu Lys Ile Ala Leu                                            GAA ACA CCG GCT CGG ATC TGT CCC ATT                                            Glu Thr Pro Ala Arg Ile Cys Pro Ile                                            AAC TAC TCC CTC CTA GCC AGC CTA CTC                                            Asn Tyr Ser Leu Leu Ala Ser Leu Leu                                            CCA AAA GGA TAC CCC GGC CGG GTG AAT                                            Pro Lys Gly Tyr Pro Gly Arg Val Asn                                            GAA ATT TTA CAC ATA CTC ATC CAA ACC                                            Glu Ile Leu His Ile Leu Ile Gln Thr                                            CAA GCC CAG ATC CCG TCC CGT CCC GCG                                            Gln Ala Gln Ile Pro Ser Arg Pro Ala                                            CCA CCG CCG CCG TCA TCC CCC ACC CAC                                            Pro Pro Pro Pro Ser Ser Pro Thr His                                            GAC CCC CCG GAT TCT GAT CCA CAA ATC                                            Asp Pro Pro Asp Ser Asp Pro Gln Ile                                            CCC CCT CCC TAT GTT GAG CCT ACG GCC                                            Pro Pro Pro Tyr Val Glu Pro Thr Ala                                            CCC CAA GTC CTT CCA GTC ATG CAT CCA                                            Pro Gln Val Leu Pro Val Met His Pro                                            CAT GGT GCT CCT CCT AAC CAT CGC CCA                                            His Gly Ala Pro Pro Asn His Arg Pro                                            TGG CAA ATG AAA GAC CTA CAG GCC ATT                                            Trp Gln Met Lys Asp Leu Gln Ala Ile                                            AAG CAA GAA GTC TCC CAA GCA GCC CCT                                            Lys Gln Glu Val Ser Gln Ala Ala Pro                                            GGG AGC CCC CAG TTT ATG CAG ACC ATC                                            Gly Ser Pro Gln Phe Met Gln Thr Ile                                            CGG CTT GCG GTG CAG CAG TTT GAC CCC                                            Arg Leu Ala Val Gln Gln Phe Asp Pro                                            ACT GCC AAA GAC CTC CAA GAC CTC CTG                                            Thr Ala Lys Asp Leu Gln Asp Leu Leu                                            CAG TAC CTT TGC TCC TCC CTC GTG GCT                                            Gln Tyr Leu Cys Ser Ser Leu Val Ala                                            TCC CTC CAT CAC CAG CAG CTA GAT AGC                                            Ser Leu His His Gln Gln Leu Asp Ser                                            CTT ATA TCA GAG GCC GAA ACC CGA GGT                                            Leu Ile Ser Glu Ala Glu Thr Arg Gly                                            ATT ACA GGT TAT AAC CCA TTA GCC GGT                                            Ile Thr Gly Tyr Asn Pro Leu Ala Gly                                            CCC CTC CGT GTC CAA GCC AAC AAT CCA                                            Pro Leu Arg Val Gln Ala Asn Asn Pro                                            CAA CAA CAA GGA TTA AGG CGA GAA TAC                                            Gln Gln Gln Gly Leu Arg Arg Glu Tyr                                            CAG CAA CTC TGG CTC GCC GCC TTC GCC                                            Gln Gln Leu Trp Leu Ala Ala Phe Ala                                            GCC CTG CCG GGG AGT GCC AAA GAC CCT                                            Ala Leu Pro Gly Ser Ala Lys Asp Pro                                            TCC TGG GCC TCT ATC CTC CAA GGC CTG                                            Ser Trp Ala Ser Ile Leu Gln Gly Leu                                            GAG GAG CCT TAC CAC GCC TTC GTA GAA                                            Glu Glu Pro Tyr His Ala Phe Val Glu                                            CGC CTC AAC ATA GCT CTT GAC AAT GGG                                            Arg Leu Asn Ile Ala Leu Asp Asn Gly                                            CTG CCA GAA GGC ACG CCC AAA GAC CCC                                            Leu Pro Glu Gly Thr Pro Lys Asp Pro                                            ATC TTA CGT TCC TTA GCC TAC TCC AAT                                            Ile Leu Arg Ser Leu Ala Tyr Ser Asn                                            GCA AAC AAA GAA TGC CAA AAA TTA CTA                                            Ala Asn Lys Glu Cys Gln Lys Leu Leu                                            CAG GCC CGA GGA CAC ACT AAT AGC CCT                                            Gln Ala Arg Gly His Thr Asn Ser Pro                                            CTA GGA GAT ATG TTG CGG GCT TGT CAG                                            Leu Gly Asp Met Leu Arg Ala Cys Gln                                            ACC TGG ACC CCC AAA GAC AAA ACC AAA                                            Thr Trp Thr Pro Lys Asp Lys Thr Lys                                            GTG TTA GTT GTC CAG CCT AAA AAA CCC                                            Val Leu Val Val Gln Pro Lys Lys Pro                                            CCC CCA AAT CAG CCG TGC TTC CGG TGC                                            Pro Pro Asn Gln Pro Cys Phe Arg Cys                                            GGG AAA GCA GGC CAC TGG AGT CGG GAC                                            Gly Lys Ala Gly His Trp Ser Arg Asp                                            TGC ACT CAG CCT CGT CCC CCC CCC GGG                                            Cys Thr Gln Pro Arg Pro Pro Pro Gly                                            CCA TGC CCC CTA TGT CAA GAC CCA ACT                                            Pro Cys Pro Leu Cys Gln Asp Pro Thr                                            CAC TGG AAG CGA GAC TGC CCC CGC CTA                                            His Trp Lys Arg Asp Cys Pro Arg Leu                                            AAG CCC ACT ATC CCA GAA CCA GAG CCA                                            Lys Pro Thr Ile Pro Glu Pro Glu Pro                                            GAG GAA GAT GCC CTC CTA TTA GAC CTC                                            Glu Glu Asp Ala Leu Leu Leu Asp Leu                                            CCC GCT GAC ATC CCA CAC CCA AAA AAC                                            Pro Ala Asp Ile Pro His Pro Lys Asn                                            TCC ATA GGG GGG GAG GTT                                                        Ser Ile Gly Gly Glu Val                                                        __________________________________________________________________________

From Table 1 above, it is shown that the precursor of core proteins is composed of 429 amino acids. In light of the structure of the terminus p-24 previously reported (Proc. Natl. Acad. Sci., U.S.A., vol 79, pp. 1291-1294 (1982)), it was expected that the precursor would be further cleaved to form core proteins having the termini p-14, p-24 and p-10.

Based on the foregoing viewpoint, the present inventors have found specific peptides which can be haptens of proteins (core proteins) associated with human leukemia virus described above and have accomplished the present invention.

SUMMARY OF THE INVENTION

That is, the present invention relates to an antibody of a human leukemia virus-related peptide obtained by collecting an antibody produced in a mammal body by administering to the mammal an antigen prepared by reacting a human leukemia virus-related peptide selected from the group consisting of:

(a) a peptide represented by formula (1):

    H-Tyr-Val-Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu-OH               (1)

(b) a peptide represented by general formula (2):

    R-Ile-Pro-His-Pro-Lys-Asn-Ser-Ile-Gly-Gly-Glu-Val-OH       (2)

wherein R is a hydrogen atom or a group represented by the general formula H-Tyr;

(c) a peptide represented by general formula (3):

    R-Thr-Trp-Thr-Pro-Lys-Asp-Lys-Thr-Lys-Val-Leu-OH           (3)

wherein R is the same as defined above;

(d) a peptide represented by formula (4):

    H-Val-Val-Gln-Pro-Lys-Lys-Pro-Pro-Pro-Tyr-OH               (4)

(e) a peptide represented by general formula (5):

    R-Met-Gly-Gln-Ile-Phe-Ser-Arg-Ser-Ala-Ser-Pro-OH           (5)

wherein R is the same as defined above; and,

(f) a peptide represented by formula (6):

    H-Tyr-Pro-Glu-Gly-Thr-Pro-Lys-Asp-Pro-Ile-leu-Arg-Ser-Leu-OH (6)

as a hapten, with a carrier in the presence of a hapten-carrier binding agent.

The present invention further relates to a process for production of the foregoing antibody.

The present invention also relates to human leukemia virus-related peptide and a process for production thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 3 are curves showing the reactivity (specificity) of the antibody of the present invention to ATL-associated antigen (ATLA) and Peptide A, D or F.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The peptides of the present invention shown by formulae (1) to (6) described above can all be easily prepared by simple operations utilizing easily accessible, commercially available amino acids. From each of the peptides, antigens can be prepared using them as haptens. From the thus obtained antigens, antibodies having a specific reactivity with virus-associated proteins can be obtained. These specific antibodies are usable for purification of virus-associated proteins, by binding these antibodies to carriers for use of, e.g., affinity chromatography, and utilizing the bound antibodies in the chromatography, etc. The specific antibodies can also be utilized as specific antibodies in various immunological measurements of such virus-associated proteins. Thus, these antibodies are useful for diagnosis of human leukemia virus infections and further for diagnosis, studies, etc. of mature T-cell leukemia or lymphoma such as adult T-cell leukemia, cutaneous T-cell lymphoma, etc. as well as diseases related thereto.

The peptides of the present invention represented by the general formulae (1) through (6) can be prepared by conventional processes for synthesizing peptides; more specifically, using processes as described in Schroder and Luhke, The Peptides, vol. 1 (1966), published by Academic Press, New York, U.S.A., or Izumiya et al., Synthesis of Peptides, (1975), published by Maruzen Publishing Co., Ltd., for example, an azide process, a chloride procees, an acid anhydride process, a mixed anhydride process, a DCC process, an active ester process (a p-nitrophenyl ester process, an N-hydroxysuccinimide ester process, a cyanomethyl ester process, etc.), a process using a Woodward reagent K, a carbodiimidazole process, an oxidative reduction process, a DCC/additive (HONB, HOBt, HOSu) process, etc. Solid phase and liquid phase syntheses are both applicable to the foregoing processes.

The peptides of the present invention are prepared in accordance with the aforesaid processes for synthesizing ordinary polypeptides, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid one by one in sequence, or by coupling fragments divided into several groups to the terminal amino acid. In more detail, for example, in case that a solid phase synthesis is adopted, the C terminal amino acid is bound to an insoluble carrier through its carboxyl group. The insoluble carrier is not particularly limited as long as it has a binding capability to a reactive carboxyl group. Examples of such insoluble carriers include halogenomethyl resins such as chloromethyl resin, bromomethyl resin, etc.; hydroxymethyl resins, phenol resins, tert-alkyloxycarbonylhydrazidated resins, etc.

After the amino protective group is removed, an amino group-protected amino acid is bound in sequence in accordance with the amino acid sequence shown by general formulae (1) through (6) through condensation of its reactive amino group and the reactive carboxyl group, in sequence, to synthesize step by step. After synthesizing the complete sequence, the peptide is split off from the insoluble carrier to produce the protein.

In the foregoing process, it is preferred that respective amino acids of histidine, arginine, tyrosine, glutamic acid, threonine, lysine, aspartic acid and serine be protected at the side chain functional groups. These functional groups at the side chain are protected with ordinary protective groups which are split off after completion of the reaction. The functional groups which take part in the reaction are generally activated. These processes are known and reagents used in these processes are also appropriately chosen from known ones.

Examples of protective groups for amino groups include a benzyloxycarbonyl, Boc, tert-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, Cl-Z, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, o-nitrophenylsulfenyl, diphenylphosphinothioyl group, etc.

Examples of protective groups for the imino group of histidine include a Tos, Bzl, benzyloxycarbonyl, trityl group, etc.

Examples of protective groups for arginine include a Tos, nitro, benzyloxycarbonyl group, etc.

Examples of protective groups for the hydroxy groups of serine and threonine include a Bzl, tert-butyl, acetyl, tetrahydropyranyl group, etc.

Examples of protective groups for the hydroxy group of tyrosine include a Bzl, Cl₂ -Bzl, benzyloxycarbonyl, acetyl, Tos group, etc.

Examples of protective groups for the amino group of lysine include a benzyloxycarbonyl, Cl-Z, Cl₂ -Bzl, Boc, Tos group, etc.

Protection for the carboxyl groups of glutamic acid and aspartic acid includes esterification of the carboxylic acids with benzyl alcohol, methanol, ethanol, tert-butanol, etc.

Examples of activated carboxyl groups include the corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (esters with pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-dicarboxydiimide, etc.).

In some cases, the peptide bond forming reaction may also be carried out in the presence of carbodiimide reagents such as dicyclohexylcarbodiimide, carbodiimidazole, etc. or tetraethylpyrophosphine, etc.

Hereafter, the preparation of the peptides in accordance with the present invention will be explained more specifically with reference to reaction equations below, as an example. ##STR1## wherein A represents a protective group for an amino group and R¹ represents an insoluble carrier.

Of the foregoing, preferred examples of A include Boc, a benzyloxycarbonyl group, a p-methoxybenzyloxycarbonyl group or the like and preferred examples of R¹ include chloromethylated polystyrene or the like, respectively.

In case that amino acids used possess functional groups at the side chain thereof which do not participate in each of the reactions, the amino acids are protected by the protective groups described above in a conventional manner and the protective groups are split off at the same time as splitting-off of the insoluble carrier R¹.

In the processes described above, the reaction of the amino acid (a) with the insoluble carrier R¹ is carried out by utilizing the reactive carboxyl group of the amino acid (a) and binding it to R¹ in a conventional manner. The reaction is effected in an appropriate solvent in the presence of basic compounds, e.g., triethylamine, potassium tert-butoxide, cesium carbonate, cesium hydroxide, etc., in the case of using, e.g., chloromethylated polystyrene. Examples of solvents include dimethylformamide (DMF), dimethylsulfoxide (DMSO), pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, N-methylpyrrolidone, hexamethylphosphoric acid triamide, etc. or a mixture solvent thereof. The above reaction is generally completed at temperatures of about 0° to about 85° C., preferably at 25° to 80° C. for several minutes to about 24 hours. It is preferred that an amount of the amino acid to the insoluble carrier be set forth such that the former is employed in an excess amount, generally 1 to 3 time equivalents per equivalent of the latter.

Splitting of the protective group A for the thus obtained amino acid shown by general formula (b) is carried out in a conventional manner. For example, there are hydrogenation using catalysts such as palladium, palladium black, etc.; a reductive method such as reduction with metallic sodium in liquid ammonia; acidolysis using strong acids such as trifluoroacetic acid, hydrogen chloride, hydrogen fluoride, methanesulfonic acid, hydrogen bromide, etc. The hydrogenation using the foregoing catalysts can be carried out, e.g., under hydrogen pressure of 1 atm at temperatures of 0° to 40° C. It is preferred that the catalyst be used generally in an amount of about 100 mg to about 1 g. The reaction is generally completed within about 1 to about 48 hours. The acidolysis described above is carried out generally at temperatures of about 0° to about 30° C., preferably 0° to 20° C. for about 15 minutes to about 1 hour, in the absence of any solvent. It is preferred that the acid be used in an amount of generally 5 to 10 times that of the raw compound. When splitting-off of the protective group A alone is desired in the acidolysis, it is preferred to use trifluoroacetic acid or hydrogen chloride as the acid. The aforesaid reduction with metallic sodium in liquid ammonia can be carried out generally at temperatures of about -40° to about -70° C., using metallic sodium in such an amount that the reaction mixture is colored to permanent blue for about 30 seconds to about 10 minutes.

The reaction of th subsequently obtained amino acid in a solid phase shown by general formula (c) and the amino acid (d) (or a derivative thereof in which the carboxyl group is activated) is carried out in the presence of a solvent. As solvents, there can be used various known solvents conventionally used in peptide condensation, for example, anhydrous dimethylformamide, dimethylsulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, ethyl acetate, N-methylpyrrolidone, hexamethylphosphoric acid triamide or a solvent mixture thereof. The reaction can also be conducted, if necessary and desired, in the presence of reagents conventionally employed in ordinary peptide bond forming reactions, for example, dehydrating and condensing agents such as carbodiimides, e.g., N,N-dicyclohexylcarbodiimide (DCC), N-ethyl-N'-dimethylaminocarbodiimide, 1-ethyl-3-diisopropylaminocarbodiimide, 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide, etc. While there is no particular limitation to the proportion of the amino acid (c) to the amino acid (d) to be used, it is preferred that the latter be employed in an amount of an equimolar amount to 10 time moles that of the former, preferably from an equimolar amount to 5 time moles. There is no particular limitation to the amount of the dehydrating and condensing agent to be used, either; the agent is generally employed preferably in an equimolar amount to that of the amino acid (d). The reaction temperature is suitably chosen from a normal range conventionally used for peptide bond forming reactions, generally from the range of about -40° to about 60° C., preferably from the range of about -20° to about 40° C. The reaction time is generally set forth for about several minutes to about 30 hours.

The thus obtained peptide shown by general formula (e) is, after splitting-off the protective group A as described above, condensed in sequence with each of the amino acids, A-Pro-OH, A-Pro-OH, A-Lys-OH, A-Lys-OH, A-Pro-OH, A-Gln-OH, A-Val-OH and A-Val-OH, in accordance with the amino acid sequence shown by general formula (4) or, derivatives thereof wherein the functional groups at the side chain are protected or the carboxyl groups are activated. Thus, the peptide shown by general formula (e) can be introduced into the peptide represented by general formula (f). These condensation and splitting-off of the protective group A are carried out in a manner similar to those described above.

The thus obtained peptide (f) can be introduced into the peptide shown by general formula (4) by splitting-off of the protective group A, splitting-off of the protective groups of the amino acid at the side chain thereof and removing the insoluble carrier R¹. Here the removal of the protective groups at the side chain functional groups and the insoluble carrier R¹ can be carried out in a manner similar to the splitting-off of the protective group A; in this case, it is preferred to use hydrogen fluoride or hydrogen bromide as the acid. All of the amino acids used in the aforesaid processes may be those commercially available.

The thus produced peptide of the present invention shown by formula (4) can be isolated and purified from the reaction mixture by means of peptide separation, e.g., extraction, distribution, column chromatography, etc.

Further, the peptides represented by general formulae (1) to (3), (5) and (6) can also be prepared in a manner similar to the process described above.

The thus obtained peptides of the present invention are utilizable as labelled antigens employed in radioimmunoassay (RIA) or enzyme immunoassay (EIA), by introducing thereto radioactive substances such as ¹²⁵ I, ¹³¹ I, etc.; various enzyme reagents such as peroxidase (POX), chymotripsinogen, procarboxypeptidase, glyceraldehyde-3-phosphatedehydrogenase, amylase, phosphorylase, D-Nase, P-Nase, β-galactosidase, glucose-6-phosphate dehydrogenase, ornithine decarboxylase, etc. The introduction of the above radioactive substance can be effected in a conventional manner. For example, the introduction of radioactive iodine can be carried out by the oxidative iodination method using chloramine T, (W. M. Hunter and F. C. Greenwood, Nature, 194, 495 (1962), Biochem. J., 89, 144 (1963)), etc. The introduction of enzyme reagents can be conducted by known methods such as conventional coupling reactions, e.g., the B. F. Erlanger, et al method (Acta Endocrinol. Suppl., 168, 206 (1972)), the M. H. Karol et al method (Proc. Natl. Acd. Sci. U.S.A., 57, 713 (1967)), etc.

Hereafter processes for production of antigens using the peptides of the present invention as haptens will be described in detail.

The aforesaid antigens are prepared by using the peptides of the present invention as haptens and reacting the peptides with a suitable carrier in the presence of a hapten-carrier binding agent. In this case, natural and synthetic proteins having a high molecular weight which are conventionally employed in the preparation of antigens can be widely employed as carriers to be bound to haptens. Examples of such carriers include albumins of animal sera such as horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, sheep serum albumin, etc.; globulins of animal sera such as horse serum globulin, bovine serum globulin, rabbit serum globulin, human serum globulin, sheep serum globulin, etc.; thyroglobulins of animals such as horse thyroglobulin, bovine thyroglobulin, rabbit thyroglobulin, human thyroglobulin, sheep thyroglobulin, etc.; hemoglobulins of animals such as horse hemoglobulin, bovine hemoglobulin, rabbit hemoglobulin, human hemoglobulin, sheep hemoglobulin, etc.; hemocyanins of animals such as Keyhole limpet hemocyanin (KLH), etc.; proteins extracted from ascaris (ascaris extracts, those described in Japanese Patent Application (OPI) No. 16414/81, J. Immun., 111, 260-268 (1973), ibid., 122, 302-308 (1979), ibid., 98, 893-900 (1967) and Am. J. Physiol., 199, 575-578 (1960), or purified products thereof); polylysine, polyglutamic acid, lysine-glutamic acid copolymers, copolymers containing lysine or ornithine, etc.

As hapten-carrier binding agents, those conventionally employed in the preparation of antigens can be widely employed. Specific examples of these agents include diazonium compounds for cross linking tyrosine, histidine, tryptophane, etc., e.g., bisdiazotized benzidine (BDB), bisdiazotized-3,3'-dianisidine (BDD), etc.; aliphatic dialdehydes for cross linking an amino group with an amino group, e.g., glyoxal, malonedialdehyde, glutaraldehyde, succinaldehyde, adipaldehyde, etc.; dimaleimide compounds for cross linking a thiol group with a thiol group, e.g., N,N'-o-phenylenedimaleimide, N,N'-m-phenylenedimaleimide, etc.; maleimidocarboxyl-N-hydroxysuccinimide esters for cross linking an amino group with a thiol group, e.g., metamaleimidobenzoyl-N-hydroxysuccinimide ester, 4-(maleimidomethyl)-cyclohexane-1-carboxyl-N'-hydroxysuccinimide ester, etc.; agents used in conventional peptide bond forming reactions in which amide bonds are formed from an amino group and a carboxyl group, e.g., dehydrating and condensing agents such as carbodiimides, e.g., N,N-dicyclohexylcarbodiimide, N-ethyl-N'-dimethylaminocarbodiimide, 1-ethyl-3-diisopropylaminocarbodiimide, 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide, etc. As the foregoing hapten-carrier binding agent, it is also possible to use diazonium aryl carboxylic acids such as p-diazonium phenylacetic acid, etc. with conventional peptide bond forming agents such as the dehydrating and condensing agents described above in combination.

The reaction for preparing the antigens described above is carried out in an aqueous solution or a conventional buffer solution having pH of 7 to 10, preferably in a buffer solution having pH of 8 to 9, at temperatures of about 0° to 40° C., preferably around room temperature. The reaction is generally completed within about 1 to about 24 hours, preferably 3 to 5 hours. Representative examples of buffer solutions which can be used in the above process include:

0.2N sodium hydroxide-0.2M boric acid-0.2M potassium chloride buffer solution

0.2M sodium carbonate-0.2M boric acid-0.2M potassium chloride buffer solution

0.05M sodium tetraborate-0.2M boric acid-0.05M sodium chloride buffer solution

0.1M dihydrogen potassium phosphate-0.05M sodium tetraborate buffer solution

In the above, proportions of the hapten, hapten-carrier binding agent and carrier can be appropriately determined but it is preferred that the carrier be employed in an amount of about 1 to about 6 times, preferably about 1 to about 5 times and the hapten-carrier binding agent be employed in an amount of about 5 to about 10 times, the weight of the hapten. By the above reaction, the carrier is bound to the hapten via the hapten-carrier binding agent to obtain a desired antigen composed of a peptide-carrier complex.

After completion of the reaction, the thus obtained antigen can easily be isolated and purified by means of a dialysis method, a gel filtration method, a fractionation precipitation method, etc.

The thus obtained antigen binds 5 to 60 mols in average of the peptide thereto per mole of a protein and enables one to subsequently prepare of an antibody having a high specificity to the antigen.

The preparation of an antibody using the antigen is carried out by administering the aforesaid antigen to mammals to thereby produce a desired antibody in vivo and collecting the antibody.

While there is no particular limitation to mammals provided for the preparation of antibodies, it is generally preferred to use rabbits or guinea pigs. In the production of antibodies, a definite amount of the antigen obtained as described above is diluted with a physiological saline solution to a suitable concentration and the resulting dilution is mixed with a complete Freund's adjuvant to prepare a suspension. The suspension is administered to mammals. For example, the aforesaid suspension is intracutaneously administered (1 to 5 mg/time as the amount of the antigen) to rabbit. Then the suspension is administered every two weeks over a period of 2 to 10 months, preferably 4 to 6 months to effect immunization. The collection of the antibody is carried out by collecting blood from the immunized animal after the passage of 1 to 2 weeks subsequent to the final administration, centrifuging the blood and isolating serum from the blood. According to this procedure, an antibody having an excellent specificity to the antigen used can be collected and used for assaying human leukemia virus-related proteins utilizing RIA, EIA, etc.

For purposes of explaining the present invention in more detail, preparations of the peptides shown by general formulae (1) to (6), antigens obtained from the peptides and antibodies will be shown by way of examples but the present invention is not deemed to be limited thereto.

Rf values in the respective preparation examples were measured using solvent mixtures described below by means of thin layer chromatography on silica gel.

Rf¹ . . . n-butanol-acetic acid-water (4:1:5)

Rf² . . . n-butanol-acetic acid-pyridine-water (15:3:10:12)

(Preparation of Peptides) SYNTHESIS EXAMPLE 1

(1) In 14 ml of a DMSO solution of 5.88 milliequivalents of potassium tert-butoxide 1.54 g of Boc-Leu-OH was dissolved and 5 g of chloromethylated polystyrene resin (Protein Research Promotion Foundation) was added to the solution. The mixture was reacted at 80° C. for 30 minutes. After thoroughly washing the resin, in sequence, with DMSO, 50% acetic acid/chloroform and methylene chloride, the resin was dried under reduced pressure to obtain 5.06 g of Boc-Leu-resin.

A part of the Boc-Leu-resin was hydrolyzed and subjected to amino acid analysis. The results indicate that the product contained 0.30 mmol of the amino acid/g of the resin.

(2) After washing 2.17 g of the Boc-Leu-resin obtained in (1) above three times with 30 ml of chloroform, the resin was added to 30 ml of a chloroform solution of 50% trifluoroacetic acid (TFA) and the mixture was reacted at room temperature for 20 minutes. The reaction mixture was washed once with 30 ml of chloroform, 5 times with 30 ml of methylene chloride, 3 times with 30 ml of a methylene chloride solution of 10% triethylamine and then 6 times with 30 ml of methylene chloride to obtain H-Leu-resin.

To 25 ml of a solution of 0.35 g of Boc-Val-OH in methylene chloride the H-Leu-resin described above was added and 5 ml of a solution of 0.33 g of DCC in methylene chloride was then added to the resulting mixture. The mixture was reacted at room temperature for 2 hours. After washing the resin 6 times with 30 ml of methylene chloride, the resin was added to 25 ml of a methylene chloride solution of 0.35 g of Boc-Val-OH and 0.55 g of 1-hydroxybenzotriazole. Then, 5 ml of a methylene chloride solution of 0.33 g of DCC was added thereto and the resulting mixture was again reacted in a similar manner (double coupling). The resin was thoroughly washed with methylene chloride to obtain Boc-Val-Leu-resin.

(3) In a manner similar to (2) described above, des-Boc of the Boc-Val-Leu-resin was conducted and amino acids described below were then condensed in order, each followed by conducting des-Boc.

    ______________________________________                                         Boc--Gln--ONP       0.59 g                                                     Boc--Pro--OH        0.35 g                                                     Boc--Ala--OH        0.31 g                                                     Boc--Thr(Bzl)--OH   0.50 g                                                     Boc--Pro--OH        0.35 g                                                     Boc--Glu(OBzl)--OH  0.55 g                                                     Boc--Val--OH        0.35 g                                                     Boc--Tyr(Cl.sub.2 --Bzl)--OH                                                                       0.71 g                                                     ______________________________________                                    

Thus 2.65 g of H-Tyr(Cl₂ -Bzl)-Val-Glu(OBzl)-Pro-Thr(Bzl)-Ala-Pro-Gln-Val-Leu-resin was obtained. 1.35 g of which was dissolved in 3 ml of anisole and 30 ml of hydrogen fluoride. After reacting the mixture at -20° C. for 30 minutes and then at 0° C. for 30 minutes, hydrogen fluoride was removed by distillation. The residue was extracted with 10% acetic acid and the extract was washed with ether. The aqueous layer was freeze dried and then purified by gel filtration using Sephadex G-10 (manufactured by Pharmacia Co., Ltd.; eluting liquid, 10% aqueous acetic acid solution), partition chromatography using Sephadex G-25 (manufactured by Pharmacia Co., Ltd.; eluting liquid, BuOH:AcOH:H₂ O=4:1:5) and further with LH-20 (manufactured by Pharmacia Co., Ltd.; eluting liquid, 1/1000 N-HCl) to obtain H-Tyr-Val-Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu-OH (hereafter referred to as "Peptide A").

Rf values: Rf¹ =0.12; Rf² =0.58

Elemental Analysis: as C₅₂ H₈₁ O₁₆ N₁₁.7H₂ O)

    ______________________________________                                                C (%)       H (%)   N (%)                                               ______________________________________                                         Calcd.   50.27         7.71    12.40                                           Found    50.41         7.83    12.41                                           ______________________________________                                    

Amino Acid Analysis (analyzed with Hitachi 835 Model)

    ______________________________________                                                        Analytical Data                                                 ______________________________________                                         Ala (1)              1.01                                                      Gln (1)                                                                                             2.09*                                                     Glu (1)                                                                        Leu (1)              0.99                                                      Pro (2)              2.04                                                      Thr (1)              1.04                                                      Tyr (1)              0.98                                                      Val (2)              1.84                                                      ______________________________________                                          *Detected as Glu                                                         

SYNTHESIS EXAMPLE 2

In a manner similar to Synthesis Example 1-(1) and (2), each of amino acids described below were condensed, in sequence, with 1.70 g of the Boc-Val-resin (0.296 mmol/g resin) obtained in a manner similar to Synthesis Example 1-(1) followed by des-Boc.

    ______________________________________                                         Boc--Glu(OBzl)--OH                                                                               0.43 g                                                       Boc--Gly--OH      0.22 g                                                       Boc--Gly--OH      0.22 g                                                       Boc--Ile--0H(1/2H.sub.2 O)                                                                       0.31 g                                                       Boc--Ser(Bzl)--OH 0.38 g                                                       Boc--Asn--ONP     0.46 g                                                       Boc--Lys(Cl--Z)--OH                                                                              0.53 g                                                       Boc--Pro--OH      0.28 g                                                       Boc--His(Tos)--OH 0.52 g                                                       Boc--Pro--OH      0.28 g                                                       Boc--Ile--OH(1/2H.sub.2 O)                                                                       0.31 g                                                       ______________________________________                                    

Thus, 2.25 g of H-Ile-Pro-His(Tos)-Pro-Lys(Cl-Z)-Asn-Ser(Bzl)-Ile-Gly-Gly-Glu(OBzl)-Val-resin was obtained, 0.81 g of which was mixed with 15 ml of hydrogen fluoride and 1.5 ml of anisole. The mixture was reacted at -20° C. for 30 minutes and then at 0° C. for 30 minutes. After removing an excess of hydrogen fluoride by distillation, the residue was extracted with 10% acetic acid. After washing the extract with ether, the system was freeze dried. Then, purification was conducted by gel filtration with Sephadex G-25 (1M acetic acid) and then using CM-Cellulose 23 (manufactured by Watmann Co., Ltd., 0.04M AcONH₄, pH=7.2) to obtain H-Ile-Pro-His-Pro-Lys-Asn-Ser-Ile-Gly-Gly-Glu-Val-OH (hereafter referred to as "Peptide B").

Rf value: Rf¹ =0.01; Rf² =0.42

Elemental Analysis: (as C₅₅ H₉₀ O₁₇ N₁₆.8H₂ O)

    ______________________________________                                                C (%)       H (%)   N (%)                                               ______________________________________                                         Calcd.   47.47         7.68    16.10                                           Found    47.39         7.71    15.98                                           ______________________________________                                    

Amino Acid Analysis (analyzed with Hitachi 835 Model)

    ______________________________________                                                    Analytical Data                                                     ______________________________________                                         Asn (1)       0.90*                                                            Gly (2)      1.99                                                              His (1)      1.02                                                              Ile (2)      2.02                                                              Lys (1)      1.01                                                              Pro (2)      1.99                                                              Ser (1)      0.91                                                              Val (1)      1.01                                                              Glu (1)      1.02                                                              ______________________________________                                          *Detected as Asp                                                         

SYNTHESIS EXAMPLE 3

In a manner similar to Synthesis Example 1-(2), 0.19 g of Boc-Tyr(Cl₂ -Bzl)-OH was reacted with 0.77 g of H-Ile-Pro-His(Tos)-Pro-Lys(Cl-Z)-Asn-Ser(Bzl)-Ile-Gly-Gly-Glu (OBzl)-Val-resin obtained in the same manner as in Synthesis Example 2 by double coupling. Then, the Boc group was split off with TFA to obtain 0.82 g of H-Tyr(Cl₂ -Bzl)-Ile-Pro-His(Tos)-Pro-Lys(Cl-Z)-Asn-Ser(Bzl)-Ile-Gly-Gly-Glu(OBzl)-Val-resin. The thus obtained resin was mixed with 15 ml of hydrogen fluoride and 1.5 ml of anisole. The mixture was reacted at -20° C. for 30 minutes and then at 10° C. for 30 minutes. After removing an excess of hydrogen fluoride by distillation, the residue was extracted with 10% acetic acid. After washing the extract with ether, the system was freeze dried. Then, purification was conducted by gel filtration with Sephadex G-25 (1M acetic acid) and then using CM-Sephadex G-25 (0.04M AcONH₄, pH=7.2) to obtain H-Tyr-Ile-Pro-His-Pro-Lys-Asn-Ser-Ile-Gly-Gly-Glu-Val-OH (hereafter referred to as "Peptide C").

Rf values: Rf¹ =0.02; Rf² =0.47

Elemental Analysis: (as C₆₄ H₉₉ O₁₉ N₁₇.8H₂ O)

    ______________________________________                                                C (%)       H (%)   N (%)                                               ______________________________________                                         Calcd.   49.44         7.46    15.32                                           Found    49.43         7.56    15.06                                           ______________________________________                                    

Amino Acid Analysis (analyzed with Hitachi 835 Model)

    ______________________________________                                                    Analytical Data                                                     ______________________________________                                         Asn (1)       0.89*                                                            Gly (2)      1.99                                                              Glu (1)      1.02                                                              His (1)      1.02                                                              Ile (2)      2.04                                                              Lys (1)      1.02                                                              Pro (2)      2.32                                                              Ser (1)      0.91                                                              Val (1)      1.01                                                              Tyr (1)      1.05                                                              ______________________________________                                          *Detected as Asp                                                         

SYNTHESIS EXAMPLE 4

In a manner similar to Synthesis Example 1-(1), Boc-Leu-resin was prepared. Each of amino acids shown below was reacted, in sequence, with 2.17 g of the thus obtained resin by double coupling in a manner similar to Synthesis Example 1-(2) and (3) described above. The des-Boc reaction of the Trp-containing peptide was carried out in the presence of ethanedithiol.

    ______________________________________                                         Boc--Val--OH       0.35 g                                                      Boc--Lys(Cl--Z)--OH                                                                               0.66 g                                                      Boc--Thr(Bzl)--OH  0.50 g                                                      Boc--Lys(Cl--Z)--OH                                                                               0.66 g                                                      Boc--Asp(OBzl)--OH 0.52 g                                                      Boc--Lys(Cl--Z)--OH                                                                               0.66 g                                                      Boc--Pro--OH       0.35 g                                                      Boc--Thr(Bzl)--OH  0.50 g                                                      Boc--Trp--OH       0.49 g                                                      Boc--Thr(Bzl)--OH  0.50 g                                                      ______________________________________                                    

Thus 3.01 g of H-Thr(Bzl)-Trp-Thr(Bzl)-Pro-Lys(Cl-Z)-Asp(OBzl)-Lys(Cl-Z)-Thr(Bzl)-Lys-(Cl-Z)-Val-Leu-resin was obtained. In a mixture of 15 ml of hydrogen fluoride, 1.5 ml of anisole and 0.8 ml of ethanedithiol 1.10 g of the thus obtained resin was dissolved. The solution was reacted at -20° C. for 30 minutes and then at 0° C. for 30 minutes. After removing an excess of hydrogen fluoride by distillation, the residue was extracted with 10% acetic acid. After washing the extract with ether, the system was freeze dried. Then, purification was conducted by gel filtration with Sephadex G-25 (1M acetic acid), CM-Cellulose 23 (0.05M AcONH₄, pH=7.2) and LH-20 (10⁻³ NHCl) to obtain H-Thr-Trp-Thr-Pro-Lys-Asp-Lys-Thr-Lys-Val-Leu-OH (hereafter referred to as "Peptide D").

Rf values: Rf¹ =0.01; Rf² =0.44

Elemental Analysis: (as C₈₁ H₁₀₁ O₁₇ N₁₅.17H₂ O)

    ______________________________________                                                C (%)       H (%)   N (%)                                               ______________________________________                                         Calcd.   45.15         8.39    12.95                                           Found    45.12         8.65    12.89                                           ______________________________________                                    

Amino Acid Analysis (analyzed with Hitachi 835 Model)

    ______________________________________                                                    Analytical Data                                                     ______________________________________                                         Asp (1)      0.91                                                              Lys (3)      3.20                                                              Leu (1)      1.00                                                              Pro (1)      1.00                                                              Thr (3)      2.89                                                              Trp (1)      0.93                                                              Val (1)      0.98                                                              ______________________________________                                    

SYNTHESIS EXAMPLE 5

Boc-Tyr(Cl₂ -Bzl)-OH, 0.13 g, was reacted with 0.55 g of H-Thr(Bzl)-Trp-Thr(Bzl)-Pro-Lys(Cl-Z)-Asp(OBzl)-Lys(Cl-Z)-Thr(Bzl)-Lys(Cl-Z)-Val-Leu-resin obtained in the same manner as in Synthesis Example 4 by double coupling. Then the Boc group was split off with TFA in the presence of ethanedithiol to obtain 0.59 g of H-Thr(Cl₂ -Bzl)-Trp-Thr (Bzl)-Pro-Lys(Cl-Z)-Asp(OBzl)-Lys(Cl-Z)-Thr(Bzl)-Lys(Cl-Z)-Val-Leu-resin.

The thus obtained resin was dissolved in a mixture of 15 ml of hydrogen fluoride, 1.5 ml of anisole and 0.8 ml of ethanedithiol. The solution was reacted at -20° C. for 30 minutes and then at 0° C. for 30 minutes. After removing an excess of hydrogen fluoride by distillation, the residue was extracted with 10% acetic acid. After washing the extract with ether, the system was freeze dried. Then, purification was conducted with CM-Cellulose 23 (0.05M AcONH₄, pH=7.2) and LH-20 (10⁻³ NHCl) to obtain H-Tyr-Thr-Trp-Thr-Pro-Lys-Asp-Lys-Thr-Lys-Val-Leu-OH (hereafter referred to as "Peptide E").

Rf values: Rf¹ =0.01; Rf² =0.47

Elemental Analysis: (as C₇₀ H₁₁₀ O₁₉ N₁₆.17H₂ O)

    ______________________________________                                                C (%)       H (%)   N (%)                                               ______________________________________                                         Calcd.   47.08         8.13    12.55                                           Found    46.89         8.34    12.54                                           ______________________________________                                    

Amino Acid Analysis (analyzed with Hitachi 835 Model)

    ______________________________________                                                    Analytical Data                                                     ______________________________________                                         Asp (1)      0.91                                                              Lys (3)      3.10                                                              Leu (1)      1.02                                                              Pro (1)      1.20                                                              Thr (3)      2.85                                                              Trp (1)      0.92                                                              Val (1)      0.93                                                              ______________________________________                                    

SYNTHESIS EXAMPLE 6

(1) In 42 ml of a DMSO solution of 15.33 milliequivalents of potassium tert-butoxide 7.53 g of Boc-Tyr(Cl₂ -Bzl)-OH was dissolved and 10 g of chloromethylated polystyrene resin (Protein Research Promotion Foundation) was added to the solution. The mixture was reacted at 80° C. for 30 minutes. After thoroughly washing the resin, in sequence, with DMSO, 50% acetic acid/chloroform and methylene chloride, the resin was dried under reduced pressure to obtain 12 g of Boc-Tyr(Cl₂ -Bzl)-resin.

A part of the thus obtained resin was hydrolyzed and subjected to amino acid analysis. The results indicate that the product contained 0.31 mmol of the amino acid/g of the resin.

(2) After washing 1.70 g of the Boc-Tyr(Cl₂ -Bzl)-resin obtained in (1) above three times with 30 ml of chloroform, the resin was added to 30 ml of a chloroform solution of 50% trifluoroacetic acid (TFA) and the mixture was reacted at room temperature for 20 minutes. The reaction mixture was washed once with 30 ml of chloroform, 5 times with 30 ml of methylene chloride, 3 times with 30 ml of a methylene chloride solution of 10% triethylamine and then 6 times with 30 ml of methylene chloride to obtain H-Tyr(Cl₂ -Bzl)-resin.

To 25 ml of a solution of 0.28 g of Boc-Pro-OH in methylene chloride the H-Tyr(Cl₂ -Bzl)-resin described above was added and 5 ml of a solution of 0.27 g of DCC in methylene chloride was then added to the resulting mixture. The mixture was reacted at room temperature for 2 hours. After washing the resin 6 times with 30 ml of methylene chloride the resin was added to 25 ml of a methylene chloride solution of 0.28 g of Boc-Pro-OH and 0.55 g of 1-hydroxybenzotriazole. Then, 5 ml of a methylene chloride solution of 0.27 g of DCC was added thereto and the resulting mixture was again reacted in a similar manner (double coupling). The resin was thoroughly washed with methylene chloride to obtain Boc-Pro-Tyr(Cl₂ -Bzl)-resin.

(3) In a manner similar to (2) described above, des-Boc of the Boc-Pro-Tyr(Cl₂ -Bzl)-resin was conducted. Then, amino acids, amino acids wherein functional groups at the side chain thereof were protected or carboxyl groups were activated, described below were condensed in order, followed by conducting des-Boc.

    ______________________________________                                         Boc--Pro--OH       0.28 g                                                      Boc--Pro--OH       0.28 g                                                      Boc--Lys(Cl--Z)--OH                                                                               0.55 g                                                      Boc--Lys(Cl--Z)--OH                                                                               0.55 g                                                      Boc--Pro--OH       0.28 g                                                      Boc--Gln--ONP      0.48 g                                                      Boc--Val--OH       0.29 g                                                      Boc--Val--OH       0.29 g                                                      ______________________________________                                    

Thus 2.57 g of H-Val-Val-Gln-Pro-Lys(Cl-Z)-Lys(Cl-Z)-Pro-Pro-Pro-Tyr(Cl₂ -Bzl)-resin. The thus obtained resin, 1.20 g, was dissolved in a mixture of 2 ml of anisole and 20 ml of hydrogen fluoride. The solution was incubated at -20° C. for 30 minutes and then at 0° C. for 30 minutes. Thereafter an excess of hydrogen fluoride was removed by distillation. The residue was extracted with 10% acetic acid and the extract was washed with ether. The aqueous layer was freeze dried and then purified by gel filtration using Sephadex G-25 (manufactured by Pharmacia Co., Ltd.; eluting liquid, 1M acetic acid) and further using CM-23 Cellulose (manufactued by Whatmann Co., Ltd.; eluting liquid, 0.04M ammonium acetate; pH=7.2) to obtain 162 mg of H-Val-Val-Gln-Pro-Lys-Lys-Pro-Pro-Pro-Tyr-OH (hereafter referred to as "Peptide F").

Rf values: Rf¹ =0.01; Rf² =0.27

Elemental Analysis: (as C₅₆ H₈₉ O₁₃ N₁₃.3CH₃ CO₂ H.4H₂ O)

    ______________________________________                                                C (%)       H (%)   N (%)                                               ______________________________________                                         Calcd.   53.02         7.82    12.96                                           Found    52.94         8.06    12.74                                           ______________________________________                                    

Amino acid analysis (analyzed with Hitachi 835 Model)

    ______________________________________                                                    Analytical Data                                                     ______________________________________                                         Gln (1)       1.05*                                                            Lys (2)      2.17                                                              Pro (4)      4.02                                                              Tyr (1)      1.05                                                              Val (2)      1.69                                                              ______________________________________                                          *Detected as Glu                                                         

SYNTHESIS EXAMPLE 7

In a manner similar to Synthesis Example 6-(1), Boc-Pro-resin (0.44 mmol/g resin) was prepared. Each of amino acids or derivatives thereof shown below was reacted, in sequence, with 1.20 g of the thus obtained resin by double coupling in a manner similar to Synthesis Example 6-(2) and (3) described above. The des-Boc reaction was then carried out.

    ______________________________________                                         Boc--Ser(Bzl)--OH 0.39 g                                                       Boc--Ala--OH      0.25 g                                                       Boc--Ser(Bzl)--OH 0.39 g                                                       Boc--Arg(Tos)--OH 0.56 g                                                       Boc--Ser(Bzl)--OH 0.39 g                                                       Boc--Phe--OH      0.35 g                                                       Boc--Ile--OH 1/2H.sub.2 O                                                                        0.32 g                                                       Boc--Gln--ONP     0.49 g                                                       Boc--Gly--OH      0.23 g                                                       Boc--Met--OH      0.33 g                                                       ______________________________________                                    

Thus 1.58 g of H-Met-Gly-Gln-Ile-Phe-Ser(Bzl)-Arg(Tos)-Ser(Bzl)-Ala-Ser(Bzl)-Pro-resin was obtained. In a mixture of 10 ml of hydrogen fluoride, 0.5 ml of 1,2-ethanedithiol and 1 ml of anisole of 0.40 g of the thus obtained resin was dissolved. The solution was incubated at -20° C. for 30 minutes and then at 0° C. for 30 minutes. After removing an excess of hydrogen fluoride by distillation under reduced pressure, the residue was extracted with 10% acetic acid. After washing the extract with ether, the system was freeze dried.

Then, purification was conducted by gel filtration with Sephadex G-25 (manufactured by Pharmacia Co., Ltd.; eluting liquid, 1M acetic acid) and CM-Cellulose 23 (manufactured by Whatmann Co., Ltd.; 0.1 to 0.5M ammonium acetate, pH=5.0, linear concentration gradient) to obtain 32 mg of H-Met-Gly-Gln-Ile-Phe-Ser-Arg-Ser-Ala-Ser-Pro-OH (hereafter referred to as "Peptide G").

Rf values: Rf¹ =0.01; Rf² =0.50

Elemental Analysis: (as C₅₀ H₈₁ O₁₆ N₁₅ S.CH₃ CO₂ H.5H₂ O)

    ______________________________________                                                C (%)       H (%)   N (%)                                               ______________________________________                                         Calcd.   46.94         7.20    15.79                                           Found    47.01         7.32    15.66                                           ______________________________________                                    

Amino acid analysis (analyzed with Hitachi 835 Model)

    ______________________________________                                                    Analytical Data                                                     ______________________________________                                         Ala (1)      1.07                                                              Arg (1)      1.07                                                              Gln (1)       1.00*                                                            Gly (1)      1.00                                                              Ile (1)      0.91                                                              Met (1)      0.94                                                              Phe (1)      0.93                                                              Pro (1)      1.07                                                              Ser (3)      2.98                                                              ______________________________________                                          *Detected as Glu                                                         

SYNTHESIS EXAMPLE 8

In a manner similar to Synthesis Example 6-(2) described above, 0.47 g of Boc-Tyr(Cl₂ -Bzl)-OH was reacted with 0.41 g of H-Met-Gly-Gln Ile-Phe-Ser(Bzl)-Arg(Tos)-Ser(Bzl)-Ala-Ser(Bzl)-Pro-resin by double coupling. Then, the Boc group was split off with trifluoroacetic acid to obtain H-Tyr(Cl₂ -Bzl)-Met-Gly-Gln-Ile-Phe-Ser(Bzl)-Arg(Tos)-Ser(Bzl)-Ala-Ser(Bzl)-Pro-resin. The thus obtained resin was mixed with a mixture of 10 ml of hydrogen fluoride, 1 ml of anisole and 0.5 ml of 1,2-ethanedithiol. The mixture was incubated at -30° C. for 30 minutes and then at 0° C. for 30 minutes. After removing an excess of hydrogen fluoride by distillation under reduced pressure, the residue was extracted with 10% acetic acid. After washing the extract with ether, the system was freeze dried. Then, purification was conducted by gel filtration with Sephadex G-25 (manufactured by Pharmacia Co., Ltd., 1M acetic acid) and then by HPLC using a debelogil column (manufactured by Chemco Co., Ltd.; eluting liquid, 0.1M NaH₂ PO₄ :acetonitrile=80:20). Thereafter, the purified matter was desalted with Sephadex G-25 (manufactured by Pharmacia Co., Ltd.; eluting liquid, 1M acetic acid) to obtain 9.23 mg of H-Tyr-Met-Gly-Gln-Ile-Phe-Ser-Arg-Ser-Ala-Ser-Pro-OH (hereafter referred to as "Peptide H").

Rf values: Rf¹ =0.01; Rf² =0.52

Elemental Analysis: (as C₅₉ H₉₀ O₁₈ N₁₆ S.CH₃ CO₂ H.6H₂ O)

    ______________________________________                                                C (%)       H (%)   N (%)                                               ______________________________________                                         Calcd.   48.47         7.07    14.82                                           Found    48.63         7.01    14.77                                           ______________________________________                                    

Amino acid analysis (analyzed with Hitachi 835 Model)

    ______________________________________                                                    Analytical Data                                                     ______________________________________                                         Ala (1)      1.03                                                              Met (1)      1.00                                                              Arg (1)      1.07                                                              Phe (1)      1.09                                                              Gln (1)       1.03*                                                            Pro (1)      0.99                                                              Gly (1)      1.14                                                              Ser (3)      2.68                                                              Ile (1)      1.03                                                              Tyr (1)      1.03                                                              ______________________________________                                          *Detected as Glu                                                         

SYNTHESIS EXAMPLE 9

In a manner similar to Example 6-(1), 0.27 mmol/g resin of Boc-Leu-resin was prepared. Each of amino acids or derivatives thereof described below were reacted, in sequence, with 2 g of the Boc-Leu-resin by double coupling in a manner similar to Synthesis Example 6-(2) and (3). Then des-Boc reaction followed.

    ______________________________________                                         Boc--Ser--OH        0.40 g                                                     Boc--Arg(Tos)--OH   0.58 g                                                     Boc--Leu--OH.H.sub.2 O                                                                             0.34 g                                                     Boc--Ile--OH.1/2H.sub.2 O                                                                          0.32 g                                                     Boc--Pro--OH        0.29 g                                                     Boc--Asp(OBzl)--OH  0.44 g                                                     Boc--Lys(Cl--Z)--OH 0.56 g                                                     Boc--Pro--OH        0.29 g                                                     Boc--Thr(Bzl)--OH   0.39 g                                                     Boc--Gly--OH        0.24 g                                                     Boc--Glu(OBzl)--OH  0.46 g                                                     Boc--Pro--OH        0.29 g                                                     Boc--Tyr(Cl.sub.2 --Bzl)--OH                                                                       0.57 g                                                     ______________________________________                                    

Thus 2.62 g of Boc-Tyr(Cl₂ -Bzl)-Pro-Glu(OBzl)-Gly-Thr(Bzl)-Pro-Lys(Cl-Z)-Asp(OBzl)-Pro-Ile-Leu-Arg(Tos)-Ser-Leu-resin was obtained. In a mixture of 20 ml of hydrogen fluoride and 2 ml of anisole 1.62 g of the thus obtained resin was dissolved. The solution was reacted at -20° C. for 30 minutes and then at 0° C. for 30 minutes. After removing an excess of hydrogen fluoride by distillation under reduced pressure, the residue was extracted with 10% acetic acid. After washing the extract with ether, the system was freeze dried. Then, purification was conducted by gel filtration with Sephadex G-25 (1M acetic acid) and then HPLC using debelogil column (eluting liquid, 0.1M phosphite buffer:acetonitrile=80:20) to obtain 104 mg of a peptide, H-Tyr-Pro-Glu-Gly-Thr-Pro-Lys-Asp-Pro-Ile-Leu-Arg-Ser-Leu-OH (hereafter referred to as "Peptide I").

Rf values: Rf¹ =0.01; Rf² =0.42

Elemental Analysis: (as C₇₂ H₁₁₆ O₂₂ N₁₈.CH₃ CO₂ H.7H₂ O)

    ______________________________________                                                C (%)       H (%)   N (%)                                               ______________________________________                                         Calcd.   50.16         7.62    14.23                                           Found    49.87         7.60    14.29                                           ______________________________________                                    

Amino acid analysis (analyzed with Hitachi 835 Model)

    ______________________________________                                                    Analytical Data                                                     ______________________________________                                         Asp (1)      0.96                                                              Lys (1)      1.13                                                              Arg (1)      1.02                                                              Pro (3)      2.79                                                              Glu (1)      1.05                                                              Ser (1)      0.92                                                              Gly (1)      1.03                                                              Thr (1)      0.99                                                              Ile (1)      0.99                                                              Tyr (1)      0.97                                                              Leu (2)      2.11                                                              ______________________________________                                    

SYNTHESIS EXAMPLE 10

The same procedures as in Synthesis Example 9 were repeated except that 0.58 g of Boc-Ser(Bzl)-OH was used in place of 0.40 g of Boc-Ser-OH to obtain 120 mg of Peptide I having the same physical properties as above.

Preparation of Antigen PREPARATION EXAMPLE 1

To 3.0 ml of a 0.05M phosphate buffer (pH=7.0) 5 mg of Peptide A obtained in Synthesis Example 1 and 12 mg of KLH (Sigma Co., Ltd.) were added and 0.2 ml of a 2% glutaraldehyde (GA) solution was dropwise added to the resulting solution. The mixture was stirred at room temperature for 3 hours. Thereafter the reaction mixture was dialyzed at 4° C. against distilled water overnight. After freeze drying, 16.5 mg of an antigen was obtained. The antigen is hereafter referred to as "Antigen A".

Antigen A bound thereto 10 mols of Peptide A per mol of KLH (when an average molecular weight was made 100,000) in average. This binding rate of Peptide A to the KLH was determined as follows: A fraction of Peptide A bound to the KLH was separated from another fraction of other product (dimer of Peptide A) by gel filtration of Antigen A obtained with Sephadex G-50 (eluting liquid, physiological saline solution; detection, OD 280 nm; eluting rate, 3 ml/hour; fractionated amount, 1 ml each); a calibration curve of a peptide dimer having standard concentrations was prepared to determine the amount of the aforesaid dimer; and the thus determined amount of the dimer was subtracted from the amount of Peptide A used as a raw material, assuming that the thus subtracted amount would be all bound to the KLH since neither the unreacted KLH nor Peptide A was recognized. Such is hereafter the same also in the following examples for preparing antigens.

PREPARATION EXAMPLE 2

(1) A BDB solution was prepared by adding 83.25 mg of benzidine to a solvent mixture of 20 ml of 0.2N-HCl and 3 ml of DMF, stirring the mixture under ice cooling, gradually adding 2 ml of distilled water containing 87.03 mg of sodium nitrite to the solution and then stirring the mixture for 30 minutes.

(2) In 1 ml of a 0.16M borate buffer (pH=9.0) containing 0.13M NaCl were dissolved 5.08 mg of Peptide E and 8.07 mg of KLH. The solution was slowly stirred at 4° C. To the solution 1 ml of the BDB solution obtained in (1) above was gradually added dropwise. The reaction solution was adjusted with 0.5N NaOH to pH=9.0 followed by reacting for further 2 hours at 4° C. Thereafter the reaction mixture was dialyzed at 4° C. against distilled water overnight. After freeze drying, 12.27 mg of an antigen was obtained. Hereafter the antigen is referred to as "Antigen B". Antigen B bound thereto 35 mols of Peptide E per mol of KLH in average.

PREPARATION EXAMPLE 3

In a manner similar to Preparation Example 2 described above, 12.74 mg of an antigen was obtained except for using 5.17 mg of Peptide C and 8.03 mg of KLH. Hereafter the antigen is referred to as "Antigen C". Antigen C bound thereto 42 mols of Peptide C per mol of KLH in average.

PREPARATION EXAMPLE 4

Following the preparation examples described above, antigens shown in Table 2 below were obtained.

                  TABLE 2                                                          ______________________________________                                                                    Binding                                                                               Molar Ratio of                                       Hapten    Carrier  Agent  Carrier-Hapten                               Antigen (mg)      (mg)     (mg)   Bond                                         ______________________________________                                         D       Peptide A ASC      GA     1:24                                                 (3)       (6)      (0.59)                                              E       Peptide E ASC      BDB    1:14                                                 (2)       (4)      (0.75)                                              F       Peptide A ASC      BDB    1:18                                                 (3)       (6)      (1.50)                                              G       Peptide B ASC      DCC    1:25                                                 (3)       (6)      (0.67)                                              H       Peptide C ASC      BDB    1:22                                                 (3)       (6)      (1.19)                                              I       Peptide D ASC      DCC    1:16                                                 (3)       (6)      (0.50)                                              ______________________________________                                          ASC: Ascaris extracts having an average molecular weight of 100,000.     

PREPARATION EXAMPLE 5

To 3.0 ml of a 0.05M phosphate buffer (pH=7.0) 5.09 mg of Peptide G obtained in Synthesis Example 7 and 25.10 mg of KLH were added and 0.2 ml of a 2% glutaraldehyde solution was dropwise added to the resulting solution. The mixture was stirred at room temperature for 3 hours. Thereafter the reaction mixture was dialyzed at 4° C. with distilled water overnight. After freeze drying, 26.49 mg of the desired antigen was obtained. The antigen is hereafter referred to as "Antigen J".

Antigen J bound thereto 10 mols of Peptide G per mol of KLH (when an average molecular weight was made 100,000) in average.

PREPARATION EXAMPLE 6

(1) A BDB solution was prepared by adding 83.25 mg of benzidine to a solvent mixture of 20 ml of 0.2N-HCl and 3 ml of DMF, stirring the mixture under ice cooling, gradually adding 2 ml of distilled water containing 87.03 mg of sodium nitrite to the solution and then stirring the mixture for 30 minutes.

(2) In 1 ml of a 0.16M borate buffer (pH=9.0) containing 0.13M NaCl were dissolved 5.13 mg of Peptide I and 8.10 mg of KLH. The solution was slowly stirred at 4° C. To the solution 0.5 ml of the BDB solution obtained in (1) above was gradually added dropwise. The reaction solution was adjusted with 0.5N NaOH to pH=9.0 followed by reacting for further 2 hours at 4° C. Thereafter the reaction mixture was dialyzed at 4° C. against distilled water overnight. After freeze drying, 12.76 mg of the desired antigen was obtained. Hereafter the antigen is referred to as "Antigen K". Antigen K bound thereto 18 mols of Peptide I per mol of KLH in average.

PREPARATION EXAMPLE 7

In a manner similar to Preparation Example 6 described above, 12.74 mg of an antigen was obtained except for using 5.17 mg of Peptide F and 8.03 mg of KLH. Hereafter the antigen is referred to as "Antigen L". Antigen L bound thereto 25 mols of Peptide F per mol of KLH in average.

PREPARATION EXAMPLE 8

Following the preparation examples described above, antigens shown in Table 3 below were obtained.

                  TABLE 3                                                          ______________________________________                                                                    Binding                                                                               Molar Ratio of                                       Hapten    Carrier  Agent  Carrier-Hapten                               Antigen (mg)      (mg)     (mg)   Bond                                         ______________________________________                                         M       Peptide G ASC      GA     1:19                                                 (2)       (4)      (0.37)                                              N       Peptide G ASC      DCC    1:21                                                 (2)       (4)      (0.70)                                              O       Peptide I ASC      DCC    1:17                                                 (2)       (4)      (0.57)                                              P       Peptide I ASC      GA     1:14                                                 (2)       (4)      (0.30)                                              Q       Peptide I ASC      BDB    1:13                                                 (2)       (4)      (0.69)                                              R       Peptide F ASC      BDB    1:19                                                 (5)       (10)     (2.42)                                              S       Peptide H BSA      DCC    1:22                                                 (3)       (6)      (0.40)                                              ______________________________________                                    

Preparation of Antibody PREPARATION EXAMPLE 1

After dissolving 100 μg of each of Antigens A, B and C obtained in Preparation Examples (of Antigen) 1 to 3 in 1.5 ml of a physiological saline solution, respectively, 1.5 ml of a Freund's adjuvant was added to each of the solutions to obtain suspensions. Each of the suspensions was subcutaneously administered to several rabbits (New Zealand white rabbits), respectively. Each of the suspensions was given at the same dose 6 times every 2 weeks and then at the same dose as the initial dose 3 times monthly. Seven days after the final administration, blood was collected from the test animals. Antisera were obtained by centrifugation and the desired antibodies were obtained, respectively. Antibodies are referred to as follows: "Antibody A" which was obtained from Antigen A, "Antibody B", "Antibody C", "Antibody D", "Antibody E" and "Antibody F" from Antigen B; "Antibody G", "Antibody H", "Antibody I", "Antibody J" and "Antibody K" from Antigen C.

PREPARATION EXAMPLE 2

Using each 500 μg of the antigens obtained in Preparation Example 4 of Antigen described above, antibodies shown in Table 4 below were obtained, respectively, in a manner similar to Preparation Example 1 of Antibody described above.

                  TABLE 4                                                          ______________________________________                                         Antigen No.    Antibody No.                                                    ______________________________________                                         Antigen D      Antibody L                                                                     Antibody M                                                                     Antibody N                                                      Antigen E      Antibody O                                                                     Antibody P                                                                     Antibody Q                                                      Antigen F      Antibody R                                                                     Antibody S                                                                     Antibody T                                                      Antigen G      Antibody U                                                      Antigen H      Antibody V                                                                     Antibody W                                                                     Antibody X                                                      Antigen I      Antibody Y                                                      ______________________________________                                    

PREPARATION EXAMPLE 3

After dissolving 100 μg of each of Antigens J, K and L obtained in Preparation Examples (of Antigen) 5 to 7 in 1.5 ml of a physiological saline solution, respectively, 1.5 ml of a Freund's adjuvant was added to each of the solutions to obtain suspensions. Each of the suspensions was subcutaneously administered to several rabbits (New Zealand white rabbits) (2.5 to 3.0 kg), respectively. Each of the suspensions was given to the rabbits at the same dose level 6 times every 2 weeks and then at the same dose as the initial dose 3 times monthly. Seven days after the final administration, blood was collected from the test animals. Antisera were obtained by centrifugation and the desired antibodies were obtained, respectively. Antibodies are referred to as follows: "Antibody Z" which was obtained from Antigen J; "Antibody AA", "Antibody AB", "Antibody AC", "Antibody AD", and "Antibody AE" from Antigen K; and "Antibody AF" from Antigen L.

PREPARATION EXAMPLE 4

Using each 500 μg of the antigens obtained in Preparation Example 8 of Antigen described above, antibodies shown in Table 5 below were obtained, respectively, in a manner similar to Preparation Example 3 of Antibody described above.

                  TABLE 5                                                          ______________________________________                                         Antigen No.    Antibody No.                                                    ______________________________________                                         Antigen M      Antibody AG                                                                    Antibody AH                                                     Antigen N      Antibody AI                                                                    Antibody AJ                                                     Antigen O      Antibody AK                                                                    Antibody AL                                                     Antigen P      Antibody AM                                                                    Antibody AN                                                     Antigen Q      Antibody AO                                                                    Antibody AP                                                     Antigen R      Antibody AQ                                                                    Antibody AR                                                                    Antibody AS                                                                    Antibody AT                                                                    Antibody AU                                                     Antigen S      Antibody AV                                                     ______________________________________                                    

Preparation of Labelled Peptide PREPARATION EXAMPLE 1

Peptides A, C, E, F, H and I obtained in Synthesis Examples 1, 3, 5, 6, 8 and 9, respectively were labelled in accordance with the method using chloramin T as follows:

That is, 20 μl of a 0.5M phosphate buffer containing 1 mCi of Na (¹²⁵ I) (carrier free N.E.N.) was added to 10 μl of a 0.5M phosphate buffer (pH 7.5) containing 5 μg of the aforesaid peptide and then 20 μl of a 0.5M phosphate buffer containing 20 μl of chloramin T was added thereto. After stirring the mixture for 25 seconds at room temperature, 20 μl of a 0.5M phosphate buffer containing 100 μg of sodium metabisulfite (Na₂ S₂ O₅) was added to the mixture to complete the reaction. Then, 10 μl of a cold 10% aqueous sodium iodide solution was added to the reaction mixture. The reaction mixture was passed through a Sephadex G-25 column (1.0 to 50 cm) (eluting liquid, a 0.2M ammonium acetate buffer containing 0.1% BSA and 0.01% NaN₃ ; pH 5.5) to obtain a Peptide labelled with ¹²⁵ I.

The radioactivity of each of the thus labelled peptides was not lower than 1,000 μCi/μg.

Measurement of Titer

The titer of each of the antibodies obtained as described above was measured as follows:

That is, each of the antibodies was diluted with a physiological saline solution to 10, 10², 10³, 10⁴, 10⁵, . . . times, respectively. To 100 μl each of the thus obtained dilutions, were added 0.1 ml of a labelled peptide diluted to about 9500 cpm (Labelled Peptide A in the case of Antibodies A, L to N, R to T; Labelled Peptide C in the case of Antibodies G to K and U to X; Labelled Peptide E in the case of Antibodies B to F, O to Q and Y; Labelled Peptide F in the case of Antibodies AF and AQ to AU; Labelled Peptide H in the case of Antibodies Z, AG to AJ and AV; and Labelled Peptide I in the case of Antibodies AA to AE and AK to AP, respectively) and 0.2 ml of a 0.05M phosphate buffer (pH=7.4; containing 0.25% BSA, 10 mM EDTA and 0.02% NaN₃). The mixture was incubated at 4° C. for 24 hours. The resulting antibody-¹²⁵ I-labelled peptide complex was separated from the unreacted (unbound) ¹²⁵ I-labelled peptide by the dextran-activated charcoal method and the centrifugal method (4° C., 30 minutes, 3000 rpm) and the radioactive ray was counted to measure a binding rate (%) of the antibody to the ¹²⁵ I-labelled peptide at each of the dilution concentrations. The binding rate (%) of the antibody to the ¹²⁵ I-labelled peptide is taken on the vertical axis and the dilution magnification of the antibody is taken on the abscissa. At each of the concentrations, the binding rate is plotted. Then, the dilution magnification of the antibody where the binding rate shows 50%, i.e., a titer of the antibody, is determined.

The results obtained are shown in Table 6 below.

                  TABLE 6                                                          ______________________________________                                                Antibody No.                                                                            Titer                                                          ______________________________________                                                A        5000                                                                  B         250                                                                  C        1500                                                                  D        32000                                                                 E        3400                                                                  F        5000                                                                  G        4200                                                                  H         720                                                                  I        1800                                                                  J         420                                                                  K         750                                                                  L        62500                                                                 M        16500                                                                 N        2300                                                                  O        7500                                                                  P        18500                                                                 Q        1800                                                                  R        11750                                                                 S        2500                                                                  T        3250                                                                  U        3000                                                                  V        19000                                                                 W        2500                                                                  X        9000                                                                  Y        3500                                                                  Z        1000                                                                  AA       1500                                                                  AB        50                                                                   AC       5000                                                                  AD       2500                                                                  AE       1000                                                                  AF       7500                                                                  AG        780                                                                  AH       2550                                                                  AI       15000                                                                 AJ       2700                                                                  AK        175                                                                  AL        125                                                                  AM       1000                                                                  AN       2650                                                                  AO       5250                                                                  AP       6000                                                                  AQ       2100                                                                  AR        675                                                                  AS       9500                                                                  AT       70000                                                                 AU        550                                                                  AV       10000                                                          ______________________________________                                    

Test for ATLA Specificity of Antibody:

(1) Peptide A, D or F having various concentrations and ATLA samples described below were employed as samples.

ATLA Positive Sample:

To 5×10⁹ of cultured cells of ATLA positive cell line YAM [Science, 217, pp. 737-739 (1982)] 30 ml of a physiological saline solution was added followed by homogenization. Then the mixture was centrifuged (105000×g) for 1 hour to collect the supernatant. The amount of the proteins in the supernatant was adjusted with PBS to 10 mg/ml (the amount of the proteins was measured by a coloration method using a reagent for total protein assay made by the Otsuka Assay Research Laboratories, "Tonein-TP") (hereafter the supernatant is referred to as "YAM Supernatant").

Further, a 0.05M phosphate buffer (pH 7.4) containing 0.25% BSA, 5 mM EDTA and 0.02% NaN₃ was employed as a standard diluting solution.

In each of test tubes, 0.2 ml of the standard diluting solution, 0.1 ml of a sample, 0.1 ml of Antibody L, O or AT obtained in Preparation Example (of Antibody) diluted to the corresponding final titer shown in Table 6, i.e., 62,500 for Antibody L, 7,500 for Antibody O, and 70,000 for Antibody AT, respectively, and 0.1 ml of ¹²⁵ I-labelled peptide corresponding to the above-described antibody (a dilution obtained by diluting Labelled Peptide A, E or F obtained as described above to about 10000 cpm) were charged. After incubating the mixture at 4° C. for 72 hours, 0.1 ml of normal porcine serum was added thereto. Then, 0.5 ml of a suspension of activated charcoal coated with dextran was added to the mixture. The mixture was allowed to stand for 30 minutes at 4° C. Thereafter, the mixture was centrifuged at 4° C. for 30 minutes at 3000 rpm to separate the antibody-¹²⁵ I-labelled peptide complex (B) from the unreacted (unbound) ¹²⁵ I-labelled peptide (F). The radioactive ray of the complex was counted to determine a percentage of (B) at each concentration and dilution of the respective samples. The results obtained are shown in FIGS. 1 to 3.

In each of the figures, the vertical axis represents a binding % (B/Bo×100 wherein Bo is a percentage of (B) when the concentration of a sample is made 0) and the abscissa represents concentrations of samples (concentration of Peptide A, D or F and, protein contents of YAM Supernatant).

FIG. 1 shows results of ATLA specificity test using Antibody L; curves (a) and (b) represent Peptide A and YAM Supernatant, respectively.

FIG. 2 shows results of ATLA specificity test using Antibody O; curves (a) and (b) represent Peptide D and YAM Supernatant, respectively.

FIG. 3 shows results of ATLA specificity test using Antibody AT; curves (a) and (b) represent Peptide F and YAM Supernatant, respectively.

From these figures ATLA specificity of the antibody of the present invention can be seen.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. 

What is claimed is:
 1. A human leukemia virus-related peptide selected from the group consisting of:(a) a peptide represented by general formula (1):

    H-Tyr-Val-Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu-OH               (1)

wherein the Tyr moiety may be labelled with radioactive iodine; (b) a peptide represented by the general formula (2)

    R-Ile-Pro-His-Pro-Lys-Asn-Ser-Ile-Gly-Gly-Glu-Val-OH       (2)

wherein R is a hydrogen atom or a group represented by the general formula H-Tyr, wherein the Tyr moiety may be labelled with radioactive iodine; (c) a peptide represented by the general formula (3):

    R-Thr-Trp-Thr-Pro-Lys-Asp-Lys-Thr-Lys-Val-Leu-OH           (3)

wherein R is a hydrogen atom or a group represented by the general formula H-Tyr, wherein the Tyr moiety may be labelled with radioactive iodine; (d) a peptide represented by the formula (4):

    H-Val-Val-Gln-Pro-Lys-Lys-Pro-Pro-Pro-Tyr-OH               (4)

wherein the Tyr moiety may be labelled with radioactive iodine; (e) a peptide represented by the general formula (5):

    R-Met-Gly-Gln-Ile-Phe-Ser-Arg-Ser-Ala-Ser-Pro-OH           (5)

wherein R is a hydrogen atom or a group represented by the general formula H-Tyr, wherein the Tyr moiety may be labelled with radioactive iodine; (f) a peptide represented by the formula (6):

    H-Tyr-Pro-Glu-Gly-Thr-Pro-Lys-Asp-Pro-Ile-Leu-Arg-Ser-Leu-Arg-Ser-Leu-OH (6)

wherein the Tyr moiety may be labelled with radioactive iodine.
 2. A peptide according to claim 1 which is represented by general formula (1):

    H-Tyr-Val-Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu-OH.              (1)


3. A peptide according to claim 1, which is represented by general formula (2):

    R-Ile-Pro-His-Pro-Lys-Asn-Ser-Ile-Gly-Gly-Glu-Val-OH       (2)

wherein R represents a hydrogen atom or a group represented by the general formula H-Tyr.
 4. A peptide according to claim 1, which is represented by general formula (3):

    R-Thr-Trp-Thr-Pro-Lys-Asp-Lys-Thr-Lys-Val-Leu-OH           (3)

wherein R represents a hydrogen atom or a group represented by the general formula H-Tyr.
 5. A peptide according to claim 1, which is represented by the formula (4):

    H-Val-Val-Gln-Pro-Lys-Lys-Pro-Pro-Pro-Tyr-OH.              (4)


6. A peptide according to claim 1, which is represented by general formula (5):

    R-Met-Gly-Gln-Ile-Phe-Ser-Arg-Ser-Ala-Ser-Pro-OH           (5)

wherein R represents a hydrogen atom or a group represented by the general formula H-Tyr.
 7. A peptide according to claim 1, which is represented by the formula (6):

    H-Tyr-Pro-Glu-Gly-Thr-Pro-Lys-Asp-Pro-Ile-Leu-Arg-Ser-Leu-OH. (6)


8. A peptide according to claim 1, which is represented by the formula

    H-Tyr*-Val-Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu-OH

wherein Tyr* represents Tyr labelled with radioactive iodine.
 9. A peptide according to claim 1, which is represented by the formula

    H-Tyr*-Ile-Pro-His-Pro-Lys-Asn-Ser-Ile-Gly-Gly-Glu-Val-OH

wherein Tyr* represents Tyr labelled with radioactive iodine.
 10. A peptide according to claim 1, which is represented by the formula

    H-Tyr*-Thr-Trp-Thr-Pro-Lys-Asp-Lys-Thr-Lys-Val-Leu-OH

wherein Tyr* represents Tyr labelled with radioactive iodine.
 11. A peptide according to claim 1, which is represented by the formula

    H-Val-Val-Gln-Pro-Lys-Lys-Pro-Pro-Pro-Tyr*-OH

wherein Tyr* represents Tyr labelled with radioactive iodine.
 12. A peptide according to claim 1, which is represented by the formula

    H-Tyr*-Met-Gly-Gln-Ile-Phe-Ser-Arg-Ser-Ala-Ser-Pro-OH

wherein Tyr* represents Tyr labelled with radioactive iodine.
 13. A peptide according to claim 1, which is represented by the formula

    H-Tyr*-Pro-Glu-Gly-Thr-Pro-Lys-Asp-Pro-Ile-Leu-Arg-Ser-Leu-OH

wherein Tyr* represents Tyr labelled with radioactive iodine. 